WO2011138142A1

WO2011138142A1 - Compositions and use of sulfasalazine

Info

Publication number: WO2011138142A1
Application number: PCT/EP2011/055828
Authority: WO
Inventors: Kai Johnsson; Christopher Chidley
Original assignee: Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2010-05-07
Filing date: 2011-04-13
Publication date: 2011-11-10

Abstract

The invention relates to compositions comprising sulfasalazine or sulfapyridine and products of a biosynthetic pathway that requires tetrahydrobiopterin. Such compositions are useful for the treatment of inflammatory bowel diseases or rheumatoid arthritis, but without the side effects observed with sulfasalazine alone. Furthermore the invention relates to compositions comprising sulfasalazine and 5 - aminosalicylic acid (mesalamine) for the treatment of the mentioned diseases. A further aspect of the invention is a method for monitoring the progress of sulfasalazine therapy comprising measuring the tetrahydrobiopterin level in body fluids of a patient under treatment with sulfasalazine. Yet another aspect of the invention is the use of sulfasalazine or sulfapyridine in a method of preventing pain in future treatment or in disease progression, such as delaying pain in cancer patients and preventing pain in surgery, dental care, chemotherapy or radiation therapy, preventing cardiogenic shock in acute myocardial infarction, preventing brain edema formation and intracranial hypertension in traumatic brain injury, and in a method of prevention or treatment of osteoporosis. All these various aspects of the invention are based on the observation that human sepiapterin reductase is a target of sulfasalazine and its metabolite sulfapyridine.

Description

Compositions and use of sulfasalazine

Field of the Invention The invention relates to compositions comprising sulfasalazine or its component sulfapyridine and to uses of such compositions in medical treatments based on the specific inhibition of sepiapterin reductase.

Background Art

The drug sulfasalazine 1 is used against inflammatory bowel diseases (IBDs) such as ulcerative colitis (UC) and Crohn's disease as well as against rheumatoid arthritis (RA). It was generated by linking the antibiotic sulfapyridine 2 with the anti-inflammatory 5-amino- salicylic acid (mesalamine, 3) through a diazo bond. Despite its medical importance, the mechanism of action of sulfasalazine remains obscure. Sulfasalazine is reduced by bacteria in the colon to sulfapyridine and mesalamine, and it is believed that the therapeutic effect of sulfasalazine in IBD is (partially) due to the topical release of mesalamine in the colon. However, sulfasalazine and mesalamine differ in their therapeutic properties. For example, sulfasalazine and mesalamine show similar efficacy in inducing remission of ulcerative colitis, but sulfasalazine shows superior efficacy in maintaining remission (Sutherland, L. and Macdonald, J.K., 2006, Cochrane Database Syst Rev, CD000544). Furthermore, only sulfasalazine shows efficacy against rheumatoid arthritis (Donahue, K.E. et al., 2008, Ann Intern Med 148, 124-134). The differences between sulfasalazine and mesalamine might be due to differences in the pharmaco- kinetics of the two drugs. Alternatively, sulfasalazine or sulfapyridine might have additional

Sepiapterin reductase (SPR, 261 amino acid residues) catalyzes the NADPH-dependent reduction of pyruvoyl-tetrahydropterin to tetrahydrobiopterin (BH4), which is the final step in the biosynthesis of the cofactor BH4 (Thony, B., Auerbach, G., and Blau, N., 2000, Biochem J 347 Pt 1, 1 -16). BH4 is utilized by hydroxylases that are, for example, involved in the biosynthesis of tyrosine, L-dopa, serotonin and norepinephrine. Furthermore, the cofactor is utilized by glyceryl-ether monooxygenases that generate lipids that play a role in signal transduction. Finally, all NO synthases require a tightly bound BH4 for activity. It has been demonstrated that inducible NO synthase (iNOS) is localized in the inflamed colonic epithelium in ulcerative colitis, Crohn's disease, and diverticulitis (Singer, I.I. et al., 1996, Gastroenterology 111, 871 -885). Furthermore, elevated levels of NO play an important role in rheumatoid arthritis (Farrell, A.J. et al., 1992, Ann. Rheum. Dis. 51, 1219- 1222). It has been demonstrated that BH4 is an intrinsic regulator of pain sensitivity and chronicity (Tegeder, I. et al., 2006, Nat. Med. 12, 1269-1277). Further demonstrating the role of iNOS in ulcerative colitis and rheumatoid arthritis is the fact that selective iNOS inhibitors reduced inflammation both in rheumatoid arthritis (Connor, J.R. et al., Eur J Pharmacol 273, 15-24 (1995)) and ulcerative colitis (Kankuri, E. et al., J Pharmacol Exp Ther 298, 1 128-32 (2001 )) models.

Although sulfasalazine is considered a safe drug suitable for long-term treatment, it has a number of side effects. Most frequent are headache, malaise, anorexia, nausea and vomiting (Watkinson, G., 1986, Drugs 32, Suppl 1, 1 -1 1 ). Furthermore, the use of sulfasalazine can lead to reversible oligospermia (Steeno, O.P., 1984, Eur J Obstet Gynecol Reprod Biol. 18, 361 -4). The reason for these side effects is not understood.

Summary of the Invention

The invention relates to a pharmaceutical composition comprising sulfasalazine or sulfapyridine and

(i) a product of a biosynthetic pathway that requires BH4;

(ii) a precursor of the final product of a biosynthetic pathway that requires BH4, wherein the precursor is not requiring BH4 for its conversion to the final product;

(iii) a synthetic derivative of the final product of a biosynthetic pathway that requires BH4, which is transformed to the final product of said biosynthetic pathway in the patient undergoing treatment;

(iv) a synthetic derivative of the final product of a biosynthetic pathway that requires BH4 and that can replace the final product of said biosynthetic pathway in its physiological effects;

(v) a synthetic derivative of a precursor of the final product of a biosynthetic pathway that requires BH4, which can be transformed into the final product or into a synthetic derivative of the final product that can replace the final product of this biosynthetic pathway in its physiological effects; and

(vi) a compound preventing the degradation, the reuptake or the metabolism of the product or of an intermediate for said product of a biosynthetic pathway that requires BH4.

A particular example of such a composition is a pharmaceutical composition comprising sulfasalazine plus levodopa, plus carbidopa, plus hydroxytryptophan. These

pharmaceutical compositions are used for reducing side effects of sulfasalazine treatment. A further aspect of the invention is a pharmaceutical composition comprising sulfasalazine and 5-aminosalicylic acid. This composition is useful for the efficient treatment of inflammatory bowel diseases.

A further aspect of the invention is a method for monitoring the progress of sulfasalazine therapy comprising measuring the tetrahydrobiopterin level or the serotonin level in body fluids of a patient under treatment with sulfasalazine.

A further aspect of the invention is a composition comprising sulfasalazine or sulfapyridine for use in a method of preventing pain in future treatment or in disease progression, such as delaying pain in cancer patients and preventing pain in surgery, dental care, chemotherapy or radiation therapy, or for use in a method of preventing cardiogenic shock in acute myocardial infarction, for use in a method of preventing brain edema formation and intracranial hypertension in traumatic brain injury, and for use in a method of prevention or treatment of osteoporosis.

A further aspect of the invention is a method of treatment of inflammatory bowel diseases, rheumatoid arthritis, preventing pain in future treatment or in disease progression, preventing cardiogenic shock in acute myocardial infarction, preventing brain edema formation and intracranial hypertension in traumatic brain injury, and a method of prevention or treatment of osteoporosis, which comprises administering a composition according to the invention in a quantity effective against said disease.

All these aspects of the invention are based on the observation that human sepiapterin reductase is a target of sulfasalazine and its metabolite sulfapyridine. Brief Description of the Figures

Figure 1 . Inhibition of NADPH-dependent reduction of sepiapterin to dihydrobiopterin by sepiapterin reductase (SPR). The inhibitory effect of different relevant molecules is tested based on the activity assay described in Example 2. The graph represents the normalized relative activity (relA) of SPR in function of the log-io value of the concentration of inhibitor in moles/liter (log[l]).

■ Benzylguanine-sulfasalazine

□ Sulfasalazine

▲ Sulfapyridine

• N-Acetylsulfapyridine

Δ N-Acetylserotonin

♦ Mesalamine

Figure 2. Pull-down of sepiapterin reductase (SPR) by immobilized sulfasalazine from mammalian cell extracts of cells expressing SPR with a V5 epitope tag. Sulfasalazine is immobilized by coupling its benzylguanine derivative (BG-SS, Example 1 ) to a SNAP™- GST fusion protein immobilized on glutathione sepharose beads (GE Healthcare). The pull-down is performed as described in Example 3. In the absence of BG-SS (lane 1 ) no SPR can be detected. In the presence of BG-SS (lane 2), the detection of SPR confirms the binding of BG-SS to SPR. The addition of underivatized sulfasalazine (lane 3, 1 equivalent; lane 4, 10 equivalents) disrupts the interaction.

Detailed Description of the Invention

It has now been found that human sepiapterin reductase (SPR) is a target of sulfasalazine of formula 1 :

The inhibition of recombinant SPR by sulfasalazine is determined using an activity assay (Example 2). Sulfasalazine inhibits SPR with an IC₅₀ of 25 nM. By comparison, the known SPR inhibitor N-acetyl-serotonin has an IC₅₀ of 1 1 μΜ under these conditions. The metabolites of sulfasalazine show much lower activity against SPR: Sulfapyridine inhibits SPR with an IC₅₀ of 2.5 μΜ, whereas mesalamine does not significantly inhibit SPR under these conditions. These data demonstrate that sulfasalazine 1 is a very potent inhibitor of SPR and that sulfapyridine is an inhibitor of SPR with medium potency (Figure 1 ). The inhibition of SPR and BH4 biosynthesis can also be observed in a cellular assay when total biopterin levels are measured after incubation of cells with varying concentrations of sulfasalazine and its metabolites (Example 4). In this assay, the total biopterin level is reduced from 1382 nmol/(mg total protein) measured in the absence of drug to 693 nmol/(mg total protein) after incubation with 0.1 mM sulfasalazine and to 135 nmol/(mg total protein) after incubation with 1 mM sulfasalazine. Similarly, the total biopterin level is reduced from 1382 nmol/(mg total protein) measured in the absence of drug to 383 nmol/(mg total protein) after incubation with 0.1 mM sulfapyridine and to 164 nmol/(mg total protein) after incubation with 1 mM sulfapyridine. Sulfapyridine shows higher activity in the cellular assay relative to sulfasalazine, reflecting the fact that sulfasalazine is a known substrate of efflux pumps (Dahan, A. and Amidon, G.L., 2010, Int J Pharm 386, 216-220). These data demonstrate that sulfasalazine and sulfapyridine are inhibitors of SPR and BH4 biosynthesis in vitro and in cell culture experiments.

The present invention is based on the observation that the inhibition of BH4 biosynthesis by sulfasalazine and sulfapyridine plays an important role in the mechanism of action of the drug. Depleting the level of BH4 will, for example, affect NO synthases which require the cofactor for activity. Genetic or chemical inhibition of the biosynthesis of the cofactor BH4 is known to affect the activity of NO synthase (Gross, S.S. and Levi, R., 1992, J Biol Chem 267, 25722-9; Bune, A.J. et al., 1996, Biochem Biophys Res Commun 220, 13-19; Tegeder, I. et al., 2006, Nat Med 12, 1269-77). Increased activity of NO synthase has been associated with ulcerative colitis, and chemical inhibition of NO synthase has been shown to reduce inflammation in acute experimental colitis (Kankuri, E. et al., 2001 , J Pharmacol Exp Ther 298, 1 128-32). Inhibition of SPR by sulfasalazine and sulfapyridine lowers BH4 levels, leading to reduced NO synthase activity in the gastrointestinal tract. The lowering of BH4 levels contributes to the efficacy of sulfasalazine in the treatment of inflammatory bowel diseases (IBDs). Increased NO synthase activity is also observed in rheumatoid arthritis (Farrell, A.J. et al., 1992, Ann Rheum Dis 51, 1219-22), and inhibition of BH4 biosynthesis by sulfasalazine and its metabolites results in an attenuation of that activity. BH4 by itself can also directly bind to certain other proteins and receptors, and, for example, plays a role in the biosynthesis of serotonin. A specific inhibition of BH4 synthesis through sulfasalazine opens up new therapeutic applications for this drug. Sulfasalazine is also effective in delaying pain in cancer patients and for the treatment of chronic pain. Adjunct therapy with sulfasalazine and sulfapyridine

The inhibition of the biosynthesis of BH4 is responsible for therapeutic benefits but will also contribute to side effects. For example, it is plausible that nausea, headache, anorexia and vomiting are caused by a change in the concentration of neurotransmitters such as serotonin, dopamine or norepinephrine, whose concentrations depend on BH4.

It has now been found that these side effects are circumvented and the treatment with sulfasalazine is improved by an adjunct therapy in which a compound is added to a conventional sulfasalazine or sulfapyridine therapy, which is selected from the group of compounds comprising:

(i) a product of a biosynthetic pathway that requires BH4;

The success of this strategy is supported by the observation that genetic deficiencies in sepiapterin reductase can be overcome by administration of levodopa and

hydroxytryptophan (Echenne, B., et al., 2006, Pediatr. Neurol. 35, 308-313). The invention relates to a pharmaceutical composition comprising sulfasalazine or sulfapyridine plus one or more compounds from the mentioned groups (i), (ii), (iii), (iv), (v), and/or (vi). In particular the invention relates to a pharmaceutical composition comprising

sulfasalazine plus levodopa;

sulfasalazine plus levodopa plus an L-dopa decarboxylase inhibitor such as carbidopa or benserazide;

sulfasalazine plus hydroxytryptophan;

sulfasalazine plus norepinephrine;

sulfasalazine plus epinephrine;

sulfasalazine plus melatonin;

sulfasalazine plus serotonin;

sulfasalazine plus levodopa plus a catechol-O-methyl transferase inhibitor such as entacapone;

sulfasalazine plus a norepinephrin reuptake inhibitor such as atomoxetine, mazindol, reboxetine, or viloxazine;

sulfasalazine plus a norepinephrin-dopamine reuptake inhibitor such as amineptine, bupropion, dexmethylphenidate, fencamfamine, fencamine, lefetamine, methylphenidate, pipradrol, prolintane, or pyrovalerone;

sulfasalazine plus a serotonin-norepinephrine reuptake inhibitor such as venlafaxine, desvenlafaxine, duloxetine, or milnacipran;

sulfasalazine plus a selective serotonin reuptake inhibitor such as citalopram, dapoxetine, escitalopram, fluoxetine, fluvoxamine, indalpine, paroxetine, sertraline, or zimelidine; sulfasalazine plus a tricyclic antidepressant such as amitriptyline, butriptyline,

clomipramine, dibenzepin, dosulepin, imipramine, lofepramine, nortriptyline, protriptyline, or trimipramine;

sulfasalazine plus a tetracyclic antidepressant such as amoxapine;

sulfasalazine plus a dopamine reuptake inhibitor such as amineptine, benzatropine, bupropion, dexmethylphenidate, esketamine, etybenzatropine, fencamfamine, fencamine, ketamine, lefetamine, medifoxamine, mesocarb, methylphenidate, nefopam, nomifensine, pipradrol, prolintane, pyrovalerone, tiletamine, or tripelenamine;

sulfasalazine plus an amphetamine-like drug such as lisdexamfetamine or

dextroamphetamine;

sulfasalazine plus a member of the substituted phenethylamine class such as

phenylephrine, 6-hydroxydopamine, salbutamol, β-methylphenethylamine, amphetamine, methamphetamine, ephedrine / pseudoephedrine, cathine, cathinone, methcathinone, mephedrone, ethcathinone, fenfluramine, phentermine, mescaline, octopamine, or tyramine; and

sulfasalazine plus a member of the monoamine oxidase inhibitor class such as benmoxin, hydralazine, iproclozide, iproniazid, isocarboxazid, isoniazid, mebanazine, nialamide, octamoxin, phenelzine, pheniprazine, phenoxypropazine, pivalylbenzhydrazine, procarbazine, or safrazine.

Furthermore the invention relates to a pharmaceutical composition comprising

sulfapyridine plus levodopa;

sulfapyridine plus levodopa plus an L-dopa decarboxylase inhibitor such as carbidopa or benserazide;

sulfapyridine plus hydroxytryptophan;

sulfapyridine plus norepinephrine;

sulfapyridine plus epinephrine;

sulfapyridine plus melatonin;

sulfapyridine plus serotonin;

sulfapyridine plus levodopa plus a catechol-O-methyl transferase inhibitor such as entacapone;

sulfapyridine plus a norepinephrin reuptake inhibitor such as atomoxetine, mazindol, reboxetine, or viloxazine;

sulfapyridine plus a norepinephrin-dopamine reuptake inhibitor such as amineptine, bupropion, dexmethylphenidate, fencamfamine, fencamine, lefetamine, methylphenidate, pipradrol, prolintane, or pyrovalerone;

sulfapyridine plus a serotonin-norepinephrine reuptake inhibitor such as venlafaxine, desvenlafaxine, duloxetine, or milnacipran;

sulfapyridine plus a selective serotonin reuptake inhibitor such as citalopram, dapoxetine, escitalopram, fluoxetine, fluvoxamine, indalpine, paroxetine, sertraline, or zimelidine; sulfapyridine plus a tricyclic antidepressant such as amitriptyline, butriptyline,

sulfapyridine plus a tetracyclic antidepressant such as amoxapine;

sulfapyridine plus a dopamine reuptake inhibitor such as amineptine, benzatropine, bupropion, dexmethylphenidate, esketamine, etybenzatropine, fencamfamine, fencamine, ketamine, lefetamine, medifoxamine, mesocarb, methylphenidate, nefopam, nomifensine, pipradrol, prolintane, pyrovalerone, tiletamine, or tripelenamine; sulfapyridine plus an amphetamine-like drug such as lisdexamfetamine or

dextroamphetamine;

sulfapyridine plus a member of the substituted phenethylamine class such as

sulfapyridine plus a member of the monoamine oxidase inhibitor class such as benmoxin, hydralazine, iproclozide, iproniazid, isocarboxazid, isoniazid, mebanazine, nialamide, octamoxin, phenelzine, pheniprazine, phenoxypropazine, pivalylbenzhydrazine, procarbazine, or safrazine.

Furthermore the invention relates to mixtures of the mentioned combinations, in particular to a pharmaceutical composition comprising sulfasalazine plus levodopa, plus carbidopa, plus hydroxytryptophan.

Preferred is a pharmaceutical composition comprising sulfasalazine plus levodopa; a pharmaceutical composition comprising sulfasalazine plus hydroxytryptophan; a pharmaceutical composition comprising sulfasalazine plus amineptine, benzatropine, or bupropion; and a pharmaceutical composition comprising sulfasalazine and citalopram, dapoxetine, escitalopram or fluoxetine.

Particularly preferred is a pharmaceutical composition comprising sulfasalazine plus levodopa plus an L-dopa decarboxylase inhibitor such as carbidopa or benserazide.

Most preferred is a pharmaceutical composition comprising sulfasalazine plus levodopa plus an L-dopa decarboxylase inhibitor such as carbidopa or benserazide, in particular carbidopa, plus hydroxytryptophan. Compositions for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, to warm-blooded animals, especially humans, are preferred. The compositions comprise the active ingredient(s) alone or, preferably, together with a pharmaceutically acceptable carrier. The dosage of the active ingredient(s) depends upon the disease to be treated and upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, and the mode of administration. The pharmaceutical compositions comprise from approximately 1 % to approximately 95% active ingredient or mixture of active ingredients, single-dose administration forms comprising in the preferred embodiment from approximately 20% to approximately 90% active ingredient(s) and forms that are not of single-dose type comprising in the preferred embodiment from approximately 5% to approximately 20% active ingredient(s). Unit dose forms are, for example, coated and uncoated tablets, suppositories, or capsules.

Examples are capsules containing from about 0.01 g to about 2.0 g active ingredient(s).

The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, or dissolving processes.

Suitable carriers are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropyl

methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.

Tablet cores can be provided with suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropyl- methylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient(s).

Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The hard capsules may contain the active ingredient in the form of granules, for example in admixture with fillers, such as corn starch, binders, and/or glidants, such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxy- ethylene sorbitan fatty acid ester type, may also be added.

Pharmaceutical compositions suitable for rectal administration are, for example, suppositories that consist of a combination of the active ingredient and a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.

The mentioned pharmaceutical compositions according to the invention may contain separate tablets, granules or other forms of orally acceptable formulation of the active ingredients, or may contain a mixture of active ingredients in one suitable pharmaceutical dosage form, as described above. In particular the separate orally acceptable formulations or the mixture in one suitable pharmaceutical dosage form may be slow release and controlled release pharmaceutical compositions.

Slow release and controlled release pharmaceutical compositions are preferably compositions comprising a swellable gel-like polymer, for example acrylic type polymers such as carbopol. Other suitable components of slow-release compositions are ion exchange resins, for example cation exchange resins comprising sulfonic or carboxylic acid functional groups, or also basic anion exchange resins for use with sulfasalazine with quaternary ammonium functional groups. Further slow-release pharmaceutical compositions are available by coating tablets, minitablets or granules comprising the active ingredients, either alone, mixed with swellable gel-like polymer or complexed with cation or anion exchange resins, with suitable films prepared from cellulose derivatives, e.g. ethyl cellulose and polyethylene glycol mixtures. Further slow-release pharmaceutical compositions are hydrophilic or hydrophobic matrix systems incorporating the active ingredients. Suitable matrix materials are, for example, hydroxypropyl methylcellulose (HPMC), sodium carboxy methylcellulose, alginate, polylactic acid, chitosan,

hydrogenated castor oil, ethylcellulose or methylcellulose, or also optionally substituted cyclodextrins, optionally coated with the mentioned film-forming polymers. The invention also relates to the mentioned pharmaceutical compositions as medicaments in the treatment of inflammatory bowel diseases, in particular ulcerative colitis and Crohn's disease, and in the treatment of rheumatoid arthritis. The present invention relates furthermore to a method of treatment of inflammatory bowel diseases, in particular ulcerative colitis and Crohn's disease, and of rheumatoid arthritis, which comprises administering a composition according to the invention in a quantity effective against said disease, to a warm-blooded animal requiring such treatment. The pharmaceutical compositions can be administered prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 0.01 g to approximately 5 g, preferably from approximately 0.25 g to approximately 1.5 g, of the active ingredients in a composition of the present invention.

Improved therapy in inflammatory bowel diseases (IBD) by co-administration of sulfasalazine and 5-aminosalicyclic acid.

The therapeutic activity in IBD of sulfasalazine is at least partially due to the topical release of 5-aminosalicylic acid (mesalazine, mesalamine). Mesalamine is currently also used directly in the treatments of ulcerative colitis and Crohn's disease. The present inventors have now found that sulfasalazine and sulfapyridine also have a direct therapeutic effect. The invention therefore relates to a pharmaceutical composition comprising sulfasalazine and mesalamine, and to such a pharmaceutical composition as a medicament for use in an improved treatment of ulcerative colitis and Crohn's disease. The optimum ratio of sulfasalazine to mesalamine in the inventive composition is determined by the medical doctor based on information concerning the metabolism of the drug components, and is usually within a range of 5:1 to 1 :50, preferably 1 :1 to 1 :10 (weight ratio sulfasalazine to mesalamine). The dosage will be adapted to the weight and condition of the particular patient in need of treatment and the severity of the disease, but will be lower than or equal to the dosage used in the prior art for treatment by either sulfasalazine or mesalamine.

Pharmaceutical compositions and pharmaceutical acceptable carriers used therein are described above. The pharmaceutical composition according to the invention comprising sulfasalazine and mesalamine may contain separate tablets or granules or other forms of orally acceptable formulation of sulfasalazine and mesalamine, or may contain a mixture of sulfasalazine and mesalamine in one suitable pharmaceutical dosage form. In particular, one or both of the separate orally acceptable formulations or the mixture in one suitable pharmaceutical dosage form may be a slow release pharmaceutical composition.

The invention also relates to the mentioned pharmaceutical compositions comprising sulfasalazine and mesalamine as medicaments with mesalamine formulated for rectal administration as a rectal suppository, suspension or enema.

The invention also relates to the mentioned pharmaceutical compositions comprising sulfasalazine and mesalamine as medicaments in the treatment of inflammatory bowel diseases, in particular ulcerative colitis and Crohn's disease, and in the treatment of rheumatoid arthritis.

The present invention relates furthermore to a method of treatment of inflammatory bowel diseases, in particular ulcerative colitis and Crohn's disease, and of rheumatoid arthritis, which comprises administering a composition comprising sulfasalazine and mesalamine according to the invention in a quantity effective against said disease, to a warm-blooded animal requiring such treatment. The pharmaceutical compositions can be administered prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 0.01 g to approximately 5 g, preferably from approximately 0.25 g to approximately 1 .5 g, of the active ingredients in a composition of the present invention.

Measuring tetrahydrobiopterin levels to improve the treatment with sulfasalazine

The therapeutic effect of sulfasalazine or sulfapyridine depends on the inhibition of the biosynthesis of BH4 through the inhibition of sepiapterin reductase by sulfasalazine or sulfapyridine. The efficacy of the treatment thus can be monitored by measuring BH4 levels. It is known that the levels of BH4 vary in the human population and thus different people will require different doses of sulfasalazine or sulfapyridine. The pharmacokinetic and ADME (absorption, distribution, metabolism, and excretion) properties will also vary between different patients. A further aspect of the present invention is monitoring the levels of BH4 during a sulfasalazine or sulfapyridine treatment to obtain an optimal therapeutic response but also to avoid the occurrence of side effects. Specifically, it is proposed to measure the concentrations of BH4, its precursors (7,8-dihydroneopterin, triphosphate neopterin, 6-pyruvoyl tetrahydropterin) and its metabolites (pterin, biopterin, 7,8-dihydropterin, 7,8-dihydroxanthopterin, xanthopterin, isoxanthopterin, or leucopterin) in biological samples (serum, plasma, urine, cerebrospinal fluid, synovial fluid, tissue samples). Methods to measure BH4, its precursors or its metabolites are known to those with knowledge in the art and are described in the literature (Tegeder, I. et al., 2006, Nat Med 12, 1269-1277). Another aspect of the invention is monitoring the concentration of molecules that are the products of biosynthetic pathways requiring BH4 during a sulfasalazine therapy in different tissues and samples (serum, plasma, lymph, urine, cerebrospinal fluid, synovial fluid, tissue samples) with the aim to avoid side effects, to initiate a combination therapy as described above and/or to control serotonin concentrations for the treatment of osteoporosis as described below. The compounds to be monitored include dopamine, serotonin, hydroxytryptophan, hydroxyindolacetic acid, epinephrine, nor-epinephrine, melatonin, and homovanillic acid. Methods to measure the concentrations of these molecules are well described in the literature (see for example Yoshitake, T., 2006, Biomed Chromatograph 20, 267-281 ).

The present invention relates furthermore to a method of treatment of inflammatory bowel diseases, in particular ulcerative colitis and Crohn's disease, and of rheumatoid arthritis, which comprises administering a composition comprising sulfasalazine in a quantity effective against said disease as monitored by measuring BH4 levels, to a warm-blooded animal requiring such treatment. New therapeutic applications for sulfasalazine and sulfapyridine

The mechanism of action of sulfasalazine (and sulfapyridine) involves the inhibition of sepiapterin reductase and consequently the inhibition of the biosynthesis of BH4. This is relevant for therapeutic applications in which sulfasalazine is currently used, or for which it has been proposed, in particular in the treatment of inflammatory bowel disease; stomach pain, rectal bleeding, and diarrhea caused by Crohn's disease; back pain and

inflammation spondyloarthropathies such as ankylosing spondylitis, reactive arthritis, or psoriatic arthritis; acute pouchitis, an idiopathic inflammatory condition of the ileal pouch anal anastomosis; different types of arthritis, e.g. protracted FMF arthritis; skin problems such as idiopathic urticaria, psoriasis, scleroderma, persistent alopecia areata, or lichens planus; costochondritis, anaphylaxis, inhibition of primary brain tumors; prevention of renal ischemia or reperfusion; intractable diarrhea after hematopoietic stem cell transplant; diabetic neuropathy; multiple sclerosis; as sensitizer in the chemotherapy of pancreatic cancer; obesity; inflammation and pain that follows surgery, chronic inflammatory diseases, chronic inflammatory bowel disease, osteoarthritis, osteolysis, tendonitis, sciatica, herniated discs, stenosis, myopathy, spondilothesis, lower back pain, facet pain, carpal tunnel syndrome, tarsal tunnel syndrome, failed back pain or the like, lower extremity pain, upper extremity pain, cancer, tissue pain and pain associated with injury or repair of cervical, thoracic, and/or lumbar vertebrae or intervertebral discs, rotator cuff, and articular joints; burns; Schonlein-Henoch disease; delayed pressure urticaria and angioedema, fibromyalgia; retinal damage, migraine; prevention of neuronal death from chronic or acute injuries to the CNS, glaucoma; venous insufficiency and venous ulcers; and steroid-dependent asthma.

According to the present invention, it has now been found that, since the mechanism of action of sulfasalazine relies on the inhibition of the biosynthesis of BH4, to have an optimal effect, the concentration of BH4 should be lowered prior to the event that results in pain, thereby preventing, delaying or attenuating the onset of pain.

!n particular, the invention re ates to sulfasalazine or sulfapyridine as a medicament for preventing pain, wherein the medicament is applied one or more days before a pain causing medical treatment, in particular before surgery, dental care, chemotherapy, radiation therapy, lithotripsy, removal of surgical staples, stitches, or catheter,

sclerotherapy, biopsy (especially sentinel lymph node biopsy or prostate biopsy), endoscopy, radiological procedures (such as hysterosalpingography), and cosmetic medical procedure such as dermabrasion, botox treatment, or laser hair removal.

Preferred is the use of the composition comprising sulfasalazine or sulfapyridine one, two, three, four, or five days before the pain-causing treatment, preferably one, two or three days before.

Likewise, the invention relates to sulfasalazine or sulfapyridine as a medicament for preventing pain, wherein the medicament is applied following the diagnosis of a disease which is supposed to cause pain on progression of the disease, in particular following diagnosis of cancer. Likewise, the invention relates to sulfasalazine or sulfapyridine as a medicament for preventing pain, wherein the medicament is applied prior to physical activity that will result in pain. Furthermore, the invention relates to sulfasalazine or sulfapyridine as a medicament for the treatment of neuralgia, in particular post herpetic neuralgia and trigeminal or glossopharyngeal neuralgia, arachnoiditis and neuropathy.

The present invention relates furthermore to a method of prevention of pain in a medical procedure, in particular in surgery, dental care, chemotherapy, radiation therapy, lithotripsy, removal of surgical staples, stitches, or catheter, sclerotherapy, biopsy

(especially sentinel lymph node biopsy or prostate biopsy), endoscopy, radiological procedures (such as hysterosalpingography), and cosmetic medical procedure such as dermabrasion, botox treatment, or laser hair removal, which comprises administering one o more days before the mentioned pain causing medical procedure a composition comprising sulfasalazine or sulfapyridine in a quantity preventive of pain in said medical procedure, to a warm-blooded animal requiring such treatment.

Likewise, the invention relates to a method of prevention of pain in a disease which is supposed to cause pain on progression of the disease, or prior to physical activity that will result in pain, which comprises administering a composition comprising sulfasalazine or sulfapyridine in a quantity preventive of pain in said disease progression or physical activity, to a warm-blooded animal requiring such treatment.

Furthermore, the invention relates to a method of treatment of neuralgia, in particular post herpetic neuralgia and trigeminal or glossopharyngeal neuralgia, arachnoiditis and neuropathy, which comprises administering a composition comprising sulfasalazine or sulfapyridine in a quantity effective against said disease, to a warm-blooded animal requiring such treatment. The pharmaceutical compositions can be administered prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment. In the case of an individual having a bodyweight of about 70 kg the daily dose administered is from approximately 0.01 g to approximately 5 g, preferably from approximately 0.25 g to approximately 1 .5 g, of sulfasalazine or sulfapyridine. According to the present invention, the inhibition of BH4 biosynthesis will affect the activity of NO synthases that require BH4 as a cofactor. Cardiogenic shock complicating acute myocardial infarction remains a common and lethal disorder despite aggressive use of early revascularization. Systemic inflammation, including expression of inducible nitric oxide synthase and generation of excess nitric oxide, is believed to contribute to the pathogenesis and inappropriate vasodilatation of persistent cardiogenic shock. The invention relates therefore to sulfasalazine or sulfapyridine as a medicament for the treatment of cardiogenic shock in acute myocardial infarction, optionally in combination with catecholamine neurotransmitters such as epinephrine, norepinephrine, dopamine and L-DOPA.

Traumatic brain injuries can result in brain edema and increased intercranial pressure. Inducible NO synthases are believed to play an important role in such complications. The present invention therefore relates to sulfapyridine, or to a pharmaceutical composition comprising sulfasalazine or sulfapyridine and an NO synthase inhibitor such as L-NMMA (N^G-monomethyl L-arginine) or L-NAME (N^G-nitro-L-arginine methyl ester), as a medicament for the prevention of brain edema formation and intercranial hypertension after traumatic brain injury. In particular the present invention relates to a pharmaceutical composition comprising sulfasalazine or sulfapyridine and 4-a m i n o-tet ra h yd ro b io pte ri n (known also as VAS203) as a medicament for the prevention of brain edema formation and intercranial hypertension after traumatic brain injury.

Furthermore, the invention relates io a method of treatment of cardiogenic shock in acute myocardial infarction, which comprises administering a composition comprising sulfasalazine or sulfapyridine, optionally in combination with catecholamine

neurotransmitters, in a quantity effective against said disease, to a warm-blooded animal requiring such treatment. Likewise, the invention relates to a method of prevention of brain edema formation and intercranial hypertension after traumatic brain injury, which comprises administering sulfapyridine, or a pharmaceutical composition comprising sulfasalazine or suifapyridine and an NO synthase inhibitor such as L-NMMA (N^G-monomethyl L-arginine) or L-NAME (N^G-nitro-L-arginine methyl ester), in a quantity effective against said disease, to a warmblooded animal requiring such treatment.

The mechanism of action of sulfasalazine (and sulfapyridine) involves the inhibition of sepiapterin reductase and consequently the inhibition of the biosynthesis of BH4. BH4 is a crucial cofactor for the biosynthesis of serotonin. Specifically, the hydroxylation of tryptophan to yield 5-hydroxy-tryptophan depends on the cofactor BH4.

Osteoporosis is a disease of low bone mass most often caused by an increase in bone resorption that is not sufficiently compensated for by a corresponding increase in bone formation (Rodan, G.A. and Martin, T.J., 2000, Science 289, 1508-1514). Gut-derived serotonin (GDS) inhibits bone formation (Yadav, V.K. et al., 2008, Cell 135, 825-837), and it has been demonstrated that the inhibition of serotonin biosynthesis in the gut can, prophylactically or therapeutically, be used to treat osteoporosis in ovariectomized rodents because of an isolated increase in bone formation (Yadav, V.K. et al., 2008, Nature Medicine 16, 308-312).

The present invention relates to sulfasalazine or suifapyridine as a medicament for treating, prophylactically or therapeutically, osteoporosis. Furthermore, the invention relates to a method of prevention and treatment of osteoporosis, which comprises administering sulfasalazine or sulfapyridine in a quantity effective against osteoporosis to a warm-blooded animal requiring such treatment.

Examples

Example 1 : Synthesis of benzylguanine-sulfasalazine conjugate (BG-SS) of formula 4.

Sulfasalazine (Fluka) is coupled to benzylguanine-(PEG)₄-NH₂ (Banala, S., 2008, ChemBioChem 9, 38-41 ) in one step using standard peptide coupling conditions with 1 -hydroxybenzotriazole (HOBt) and ethyl-(3-dimethylaminopropyl)carbodiimide (EDAC). The coupled product (BG-SS) is purified by reversed phase HPLC.

HRMS(ESI) m/z: [M+H]⁺ calculated for C₄iH₄₅NnOi₀S, 884.3150; found, 884.3167.

Example 2: Inhibition of NADPH-dependent reduction of sepiapterin to dihydrobiopterin. The open reading frame (ORF) of sepiapterin reductase (SPR) is inserted into vector pDEST17 (Invitrogen) for N-terminal His-tagged expression by Gateway recombination from relevant entry clones. SPR is recombinantly expressed in E.coli cells from the constructed vector and purified using Ni-NTA agarose according to the manufacturer's instructions (Qiagen). The reduction of sepiapterin to dihydrobiopterin by SPR is followed spectrophotometrically at 420 nm (SpectraMax 340). The assay is performed in 50 mM potassium phosphate pH 6.5 adjusted to 100 μΜ sepiapterin, 200 μΜ NADPH, 8 ng/μΙ SPR, 0.5% DMSO and in a volume of 200 μΙ at 25°C. The inhibitor is added from a 200x stock solution in DMSO. The reactions are started by addition of sepiapterin to a mix of all other components. Example 3: Pull-down assay.

The ORF of SPR is inserted into vector pcDNA3.1/nV5-DEST (Invitrogen) for N-terminal V5-tagged expression by Gateway recombination from relevant entry clones. U20S cells (maintained in DMEM supplemented with 10% FBS) are transfected with the resulting constructs using Lipofectamine 2000 (Invitrogen) in 3.5 cm dishes. After transient expression for 48 h, the cells are lysed on ice for 10 min in buffer A (50 mM TrisHCI pH 7.9, 150 mM NaCI, 5 mM EDTA, 1 % Triton X-100, 50 mM NaF, 1 mM Na₃V0₄, and protease inhibitor cocktail (Complete Mini, Roche). The lysate is cleared by centrifugation at 16,000 x g for 10 min at 4°C and used as input sample in subsequent pull-down experiments. For the preparation of the pull-down matrix, 20 μΙ of 20% glutathione sepharose 4B slurry (GE Healthcare) in buffer B (50 mM Tris CI pH 7.9 and 0.5 M NaCI) is mixed with an E.coli lysate containing bead saturating amounts of recombinant GST- SNAP™ for 30 min at 4°C. After washing 2x with 200 μΙ buffer A and resuspension in 50 μΙ buffer A, the immobilized GST-SNAP™ is labeled with BG-SS by addition of 0.5 μΙ of 1 mM BG-SS in DMSO. After 30 min at room temperature, the beads are washed 2x with 200 μΙ buffer A and then incubated with 50 μΙ mammalian protein extract for 1 -3 h at 4°C. After washing 3x with 200 μΙ buffer A, bound protein is eluted in 20 μΙ 50 mM TrisHCI pH 7.9 plus 10 mM reduced glutathione. Eluted proteins are resolved by SDS-PAGE and transferred to PVDF membrane. Immunoblotting is performed using anti-V5-HRP

(Invitrogen), ECL Plus Western Blotting Detection Reagents (GE Healthcare) and Kodak Image Station 440CF.

Example 4: Inhibition of tetrahydrobiopterin biosynthesis in mammalian cell culture experiments.

The effect of SPR inhibitors on cellular total biopterin levels (corresponding to the sum of biopterin, dihydrobiopterin and tetrahydrobiopterin) was analyzed in rat

pheochromatocytoma PC12 cells since these cells have been previously used for measuring the effect of SPR inhibitors on BH4 biosynthesis (Fujimoto et al., 2003, Chem Biol Interact 143-144, 583-6). Biopterin is detected using an assay based on ultra-high pressure liquid chromatography (uHPLC) and fluorescence detection. PC12 cells were maintained in DMEM GlutaMax-l (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37°C in a water-saturated 5% C0₂ incubator. Prior to incubation with relevant molecules, PC12 cells were seeded to a density of 2x10⁴ cells/cm² in cell culture flasks (25 cm²). After 48 h incubation, the growth medium was exchanged for DMEM GlutaMax-l, 10% FBS, sepiapterin 10 μΜ, tested SPR inhibitor (10 - 10Ό00 μΜ), with a final DMSO concentration of 0.2% or 1.1 %. The cells were then incubated for an additional 24 h at 37°C in the presence of the SPR inhibitor and sepiapterin (0.01 mM). The cells were harvested and washed twice with 5 ml PBS pH 7.4, and then were generally stored at -80°C until further analysis. The cells were thawed on ice and lysed in 1 .5 ml 50 mM Tris CI pH 7.4, IGEPAL (Invitrogen) 0.2%. Protein concentrations (typically 1 -2.5 μg μl) were determined in a Bradford assay using BSA as a standard. In order to measure total biopterin concentrations, dihydrobiopterin (BH2) and tetrahydrobiopterin (BH4) were oxidised to biopterin under acidic conditions as described in Nature Protocol: DOI: 10.1038/nprot.2006.298. For that purpose, 0.5 ml of a freshly prepared iodine solution (1 % (w/v) l₂, 2% (w/v) Kl in 1 M HCI) was added to 1 ml of crude cell lysate, and the samples were incubated at room temperature for 30 min in the dark. The oxidation reaction was stopped by addition of 0.2 ml of freshly prepared 5% (w/v) ascorbic acid solution. After centrifugation of the samples for 10 min at 8,800 x g, the supernatants were purified on Oasis MCX extraction cartridges (Waters). Biopterin fractions were eluted with 8% ammonia in 25% methanol. After evaporation of the solvents, the samples were recovered in H₂0/trifluoroacetic acid (TFA) 0.01 % and were filtered using a centrifugal filter device (NanoSep 100K, Pall) before liquid chromatography. The samples were analyzed by uHPLC using a SHIM-PACK XR-ODS 75Lx2.0 (Shimadzu) setup at a flow of 0.5 ml/min and using a (H₂0/TFA 0.1 %) - acetonitrile gradient. Biopterin was detected by fluorescence (excitation at 350 nm, emission at 435 nm) and was quantified using a linear calibration curve of 0.01 - 1 nmol biopterin (Sigma). Biopterin levels were normalized by the protein concentration of the cell lysates determined in a Bradford assay and are reported as nmol biopterin per mg total protein in the lysate.

Claims

1 . A pharmaceutical composition comprising sulfasalazine or sulfapyridine and

(i) a product of a biosynthetic pathway that requires BH4;

2. The pharmaceutical composition according to claim 1 comprising sulfasalazine or sulfapyridine and one or more compounds selected from the group consisting of levodopa;

levodopa plus an L-dopa decarboxylase inhibitor;

hydroxytryptophan;

norepinephrine;

epinephrine;

melatonin;

serotonin;

levodopa plus a catechol-O-methyl transferase inhibitor;

a norepinephrin reuptake inhibitor;

a norepinephrin-dopamine reuptake inhibitor;

a serotonin-norepinephrine reuptake inhibitor;

a selective serotonin reuptake inhibitor;

a tricyclic antidepressant;

a tetracyclic antidepressant such as amoxapine;

a dopamine reuptake inhibitor; an amphetamine-like drug;

a member of the substituted phenethylamine class; and

a member of the monoamine oxidase inhibitor class.

3. The pharmaceutical composition according to claim 1 comprising sulfasalazine and one or more compounds selected from the group consisting of

levodopa;

levodopa plus carbidopa or benserazide;

hydroxytryptophan;

norepinephrine;

epinephrine;

melatonin;

serotonin;

levodopa plus entacapone;

atomoxetine, mazindol, reboxetine, or viloxazine;

amineptine, bupropion, dexmethylphenidate, fencamfamine, fencamine, lefetamine, methylphenidate, pipradrol, prolintane, or pyrovalerone;

venlafaxine, desvenlafaxine, duloxetine, or milnacipran;

citalopram, dapoxetine, escitalopram, fluoxetine, fluvoxamine, indalpine, paroxetine, sertraline, or zimelidine;

amitriptyline, butriptyline, clomipramine, dibenzepin, dosulepin, imipramine, lofepramine, nortriptyline, protriptyline, or trimipramine;

amoxapine;

amineptine, benzatropine, bupropion, dexmethylphenidate, esketamine, etybenzatropine, fencamfamine, fencamine, ketamine, lefetamine, medifoxamine, mesocarb,

methylphenidate, nefopam, nomifensine, pipradrol, prolintane, pyrovalerone, tiletamine, or tripelenamine;

lisdexamfetamine or dextroamphetamine;

benmoxin, hydralazine, iproclozide, iproniazid, isocarboxazid, isoniazid, mebanazine, nialamide, octamoxin, phenelzine, pheniprazine, phenoxypropazine, pivalylbenzhydrazine, procarbazine, or safrazine.

4. The pharmaceutical composition according to claim 1 comprising sulfasalazine plus levodopa, plus carbidopa, plus hydroxytryptophan.

5. The pharmaceutical composition according to any one of claims 1 to 4 for use in reducing side effects of sulfasalazine treatment.

6. A pharmaceutical composition comprising sulfasalazine and 5-aminosalicylic acid.

7. The pharmaceutical composition according to claim 6 for use in the treatment of inflammatory bowel diseases.

8. A method for monitoring the progress of sulfasalazine or sulfapyridine therapy comprising measuring the level of tetrahydrobiopterin, its precursors 7,8-dihydroneopterin, triphosphate neopterin or 6-pyruvoyl tetrahydropterin, or its metabolites pterin, biopterin, 7,8-dihydropterin, 7,8-dihydroxanthopterin, xanthopterin, isoxanthopterin or leucopterin in body fluids or tissue samples of a patient under treatment with sulfasalazine or sulfapyridine.

9. A method according to claim 8 comprising measuring the tetrahydrobiopterin level in body fluids of a patient under treatment with sulfasalazine.

10. A method for monitoring the progress of sulfasalazine or sulfapyridine therapy comprising measuring the concentration of molecules that are the products of biosynthetic pathways requiring tetrahydrobiopterin during a sulfasalazine therapy selected from the group consisting of dopamine, serotonin, hydroxytryptophan, hydroxyindolacetic acid, epinephrine, norepinephrine, melatonin, and homovanillic acid, in body fluids or tissue samples of a patient under treatment with sulfasalazine or sulfapyridine.

1 1 . A method according to claim 10 comprising measuring the concentration of serotonin, dopamine or norepinephrine in body fluids or tissue samples of a patient under treatment with sulfasalazine or sulfapyridine.

12. A method according to claim 10 comprising measuring the serotonin level in body fluids of a patient under treatment with sulfasalazine.

13. Sulfasalazine or sulfapyridine for use in a method of preventing pain in future treatment or in disease progression.

14. Sulfasalazine or sulfapyridine according to claim 13 for use in a method of delaying pain in cancer patients.

15. Sulfasalazine or sulfapyridine according to claim 13 for use in a method of preventing pain in surgery, dental care, chemotherapy or radiation therapy.

16. Sulfasalazine or sulfapyridine for use in a method of treating neuralgia.

17. Sulfasalazine or sulfapyridine according to claim 16 for use in a method of treating post herpetic neuralgia, trigeminal or glossopharyngeal neuralgia, arachnoiditis, or neuropathy.

18. Sulfasalazine or sulfapyridine for use in a method of preventing cardiogenic shock in acute myocardial infarction.

19. A composition comprising sulfasalazine or sulfapyridine and a catecholamine neurotransmitter selected from the group consisting of epinephrine, norepinephrine, dopamine and L-DOPA for use in a method of preventing cardiogenic shock in acute myocardial infarction.

20. Sulfapyridine or sulfapyridine for use in a method of preventing brain edema formation and intracranial hypertension in traumatic brain injury.

21 . A composition comprising sulfasalazine or sulfapyridine and an NO synthase inhibitor for use in a method of preventing brain edema formation and intracranial hypertension in traumatic brain injury.

22. Sulfasalazine or sulfapyridine for use in a method of prevention or treatment of osteoporosis.